The proposed study will establish the methodology the the enzymatic isolation and the electrophoretic separation of the osteoclast precursor. The osteoclast has a central role in the skeletal process of bone resorption and it has a hematogenous origin. However, the cellular events which occur as the precursor matures to a resorbing osteoclast remain obscure. The delineation of those events would aid the development of new strategies for the treatment of skeletal disorders characterized by a high rate of bone resorption. Bone cells will be isolated from neonatal mouse calvaria (freshly dissected or cultured) using two purified enzymes from crude bacterial collagenase in a timed sequential digestion. Neonatal mouse calvaria contain resident osteoclast precursors which mature when bone resorption is stimulated with either 1,25-dihydroxycholecalciferol or parathyroid hormone. Bone cells will be isolated from calvaria in culture to take advantage of any population shifts or changes in cellular characteristics which may occur after treatment. Purified enzymes will be used to isolate cells in order to increase the cell yield and ensure the reproducibility of the cell isolation procedure. The cells isolated from each digestion period will be separated by means of free flow electrophoresis. Electrophoresis is a rapid means of cell separation and it appears to be unaffected by the proteolytic enzymes used in the cell isolation. In order to identify the precursor of the osteoclast the electrophoresed cells will be characterized by their enzymatic content, their hormonal responsiveness to parathyroid hormone and calcitonin, the presence of specific receptors for 1,25-dihydroxycholecalciferol, their ability to resorb bone, and their morphological appearance.